Influence of L-Carnitine on Post-Thaw Semen Quality in Mehsana Buffalo Bulls with Different Freezability Levels

Introduction

Male fertility is a key determinant of reproductive efficiency in bovines, particularly under artificial insemination (AI) systems where a single bull contributes to the fertilization of numerous females. Therefore, accurate evaluation of semen quality is essential for predicting fertility potential. Semen analysis remains the most reliable and widely adopted method for assessing male reproductive performance (Bisla et al., 2022; Patel et al., 2024; Patel et al., 2026) [3,17,16] . Parameters such as motility, concentration, morphology and viability are critical indicators of sperm function, and their impairment can lead to reduced conception rates and significant economic losses in the livestock sector. Hence, improving semen quality, especially during cryopreservation, is of considerable importance for enhancing breeding efficiency. L-carnitine (γ-trimethylamino-β-hydroxybutyrate), a naturally occurring compound abundant in the epididymis, plays a vital role in sperm maturation and energy metabolism by facilitating the transport of fatty acids into mitochondria for β-oxidation and ATP production. Additionally, it exhibits antioxidant properties that help mitigate reactive oxygen species (ROS)-induced damage to sperm cells (Lenzi et al., 1992; Kerner and Hoppel, 2000; Vaz and Wanders, 2002) [13,12,22] . Supplementation of L-carnitine has been reported to improve postthaw semen quality in various species, including enhanced motility in stallions, improved viability in buffaloes, and overall semen quality in cattle (Bucak et al., 2009; Gibb et al., 2015; Longobardi et al., 2017; Vaghasiya et al., 2024) [5,8,14,21] . However, its effect on semen freezability in Mehsana buffalo bulls with varying freezability levels has not been investigated. Therefore, the present study was undertaken to evaluate the effect of L-carnitine supplementation on semen quality and oxidative stress parameters in Mehsana buffalo bulls, with the objective of optimizing semen extenders and improving the efficiency of artificial insemination programs.

Materials and Methods

Grouping of Bulls

Nine healthy, sexually mature Mehsana buffalo bulls maintained at the Dama Semen Production Unit, Banas Dairy, Palanpur, were selected for the study. Based on their semen freezability, determined from one year of production records, bulls were categorized into three groups: poor (Group I), moderate (Group II), and good freezability (Group III). The classification was based on post-thaw semen straw discard rate (%), calculated as the proportion of ejaculates discarded due to less than 50% post-thaw motility. Bulls with discard rates above mean +1 SD were classified as poor, those below mean -1 SD as good, and the remaining as moderate freezability group.

Semen Collection

Prior to semen collection, preputial washing was performed using 0.9% normal saline. Semen was collected twice weekly in the morning using an artificial vagina (AV) with a male dummy, following three false mounts to ensure adequate sexual stimulation. A total of 108 ejaculates (12 per bull) were collected over a period of six weeks.

Semen Processing

Only ejaculates exhibiting initial motility ≥70% were processed further. Semen was diluted to a final concentration of 20 million spermatozoa per 0.25 ml French mini straw (TBS, IMV, France) using standard diluents. The filled and sealed straws were equilibrated at 4-5°C for 4 hours in a cold handling cabinet. Cryopreservation was carried out using a controlled freezing protocol: from 4°C to -10°C at 5°C/min, from -10°C to -100°C at 40°C/min, and from -100°C to -140°C at 20°C/min. After 24 hours of storage, straws were thawed at 37°C for 30 seconds for evaluation.

Semen Evaluation

Semen samples were assessed for individual motility, sperm viability, morphology and plasma membrane integrity at both post-dilution and post-thaw stages. Oxidative stress parameters, namely malondialdehyde (MDA) and superoxide dismutase (SOD), were evaluated at the post-thaw stage.

Statistical Analysis

Data were tested for normality using the Shapiro-Wilk test. Percentage data were arcsine transformed prior to analysis. Statistical comparisons among groups were performed using one-way ANOVA followed by Tukey's post hoc test. Results were expressed as mean ± Standard error (SE), with significance set at P<0.05. Statistical analysis was conducted using GraphPad Prism version 6.

Results and Discussion

The semen samples were examined for individual subjective motility, viability, plasma membrane integrity and sperm abnormality at post dilution and post thawed stages (Table 1). Individual subjective motility and sperm viability did not differ significantly among the three groups at the post-dilution stage. These findings are consistent with earlier reports indicating no significant effect of L-carnitine supplementation on semen quality immediately after dilution (El-Raey et al., 2016; Hussein, 2018; Vaghasiya et al., 2024). This suggests that the beneficial effects of L-carnitine may not be evident during the initial processing stage. However, at the post-thaw stage, sperm viability differed significantly (P<0.05) among the groups, with improved values observed in bulls with better freezability. These results are in agreement with previous studies reporting enhanced post-thaw sperm viability following L-carnitine supplementation ( [4, 14, 15] . These observations highlight the role of L-carnitine in maintaining membrane stability and structural integrity of spermatozoa during cryopreservation. The beneficial effects of L-carnitine can be attributed to its involvement in sperm energy metabolism and antioxidant defense. Sperm motility is largely dependent on ATP generated through mitochondrial oxidative phosphorylation. L-carnitine facilitates the transport of fatty acids into mitochondria, thereby enhancing ATP production and supporting sperm motility and survival. Additionally, it reduces oxidative stress and minimizes capacitation-like changes during cryopreservation, leading to improved postthaw semen quality (Longobardi et al., 2017) [14] . Spermatozoa are highly susceptible to oxidative damage due to the high content of polyunsaturated fatty acids in their plasma membrane. Excessive lipid peroxidation during cryopreservation can compromise membrane integrity, reduce motility and impair fertility (Griveau et al., 1995) [9] . The improved membrane integrity observed in this study suggests that L-carnitine may help protect sperm cells from such oxidative damage, thereby enhancing their functional competence after thawing. Reactive oxygen species (ROS) are naturally generated by spermatozoa and play an essential role at physiological levels in key fertilization processes such as capacitation, acrosome reaction, hyperactivation, and sperm-oocyte fusion (Ahmed et al., 2020) [1] . However, excessive ROS production during cryopreservation induces lipid peroxidation, leading to structural damage of the sperm plasma membrane and compromised sperm function (Sharma and Agarwal, 1996) [18] . The beneficial effects observed in the present study may be attributed to the multifunctional role of L-carnitine. Its membrane-stabilizing properties, along with its role in maintaining osmotic balance, contribute to preserving sperm structural integrity during freezing and thawing (Deana et al., 1984) [6] . Consequently, L-carnitine supplementation likely enhances sperm motility and increases the proportion of spermatozoa with intact plasma membranes at the post-thaw stage. The antioxidant parameter malondialdehyde (MDA) did not differ significantly among the three groups at the post-thaw stage (Table 2). These findings are consistent with earlier reports, where no significant variation in MDA concentration was observed following L-carnitine supplementation in buffalo bull semen (Bucak et [4,14,21] . This suggests that Lcarnitine supplementation at the tested levels may not markedly influence lipid peroxidation as measured by MDA. Spermatozoa are highly susceptible to lipid peroxidation due to the abundance of polyunsaturated fatty acids in their plasma membrane. During cryopreservation, excessive generation of reactive oxygen species (ROS) leads to oxidative damage, disrupting membrane integrity and impairing sperm function (Bucak et al., 2010) [4] . ROSmediated lipid peroxidation is further exacerbated by ironcatalyzed reactions such as the Haber-Weiss and Fenton reactions, which accelerate the formation of highly reactive free radicals. L-carnitine, owing to its metal-chelating and antioxidant properties, may reduce iron-induced oxidative stress by limiting free radical generation (Gulcin, 2006) [10] . Although MDA levels were not significantly altered in the present study, the observed improvements in semen quality parameters suggest a protective role of L-carnitine against oxidative damage. A similar non-significant trend was observed for superoxide dismutase (SOD) activity at the post-thaw stage. However, previous studies have reported significantly increased SOD activity following L-carnitine supplementation (MA et al., 2017; Vaghasiya et al., 2024) [15,21] . SOD is a primary antioxidant enzyme that catalyzes the dismutation of superoxide radicals into hydrogen peroxide, thereby protecting sperm cells from oxidative stress (Barranco et al., 2019) [2] . The resulting hydrogen peroxide is subsequently detoxified by other antioxidant enzymes such as catalase and glutathione peroxidase (Silvestre et al., 2021) [19] . The observed trend toward higher SOD levels in L-carnitinetreated groups may be attributed to its multifaceted antioxidant action, including ROS scavenging, metal chelation, and reduction of oxidative enzyme activity (Surai, 2015) [20] . Additionally, L-carnitine has been reported to directly neutralize reactive species such as superoxide and hydrogen peroxide (Gulcin, 2006) [10] . These combined effects may contribute to improved antioxidant defense and enhanced post-thaw sperm quality, even in the absence of statistically significant changes in oxidative stress markers.

Conclusion

L-carnitine supplementation significantly improved post-thaw sperm viability, plasma membrane integrity and morphological normality in Mehsana buffalo bull semen, with more pronounced effects observed in bulls with good freezability. These findings indicate that L-carnitine plays a beneficial role in reducing cryo-induced damage and enhancing membrane stability during semen cryopreservation in Mehsana buffalo bulls.

Acknowledgement

AcknowledgmentThe authors thank the authorities of the Banaskantha District Co-Operative Milk Producers' Union Ltd. (Banas Dairy), Palanpur, and to the laboratory staff of the Dama Semen Production Unit, Dama for providing all necessary facilities and assistance throughout the course of the research work.

References

1. Ahmed H, Jahan S, Ullah H, Ullah F, Salman MM. The addition of resveratrol in tris citric acid extender ameliorates post-thaw quality parameters, antioxidant enzymes levels, and fertilizing capability of buffalo (Bubalus bubalis) bull spermatozoa. Theriogenology. 2020;152:106-113.
2. Barranco I, Padilla L, Tvarijonaviciute A, Parrilla I, Martínez EA, Rodriguez-Martinez H, et al. Levels of activity of superoxide dismutase in seminal plasma do not predict fertility of pig AI-semen doses. Theriogenology. 2019;140:18-24.
3. Bisla A, Rautela R, Katiyar R, Kumar A, Ghosh SK, Srivastava N. Semen discard rate at fresh and post-thaw stages in Indian riverine Buffalo (Bubalus bubalis). Indian J Anim Reprod. 2022;43(1):35-42.
4. Bucak MN, Tuncer PB, Sarıozkan S, Başpınar N, Taşpınar M, Çoyan K, et al. Effects of antioxidants on post-thawed bovine sperm and oxidative stress parameters: antioxidants protect DNA integrity against cryodamage. Cryobiology. 2010;61(3):248-253.
5. Bucak MN, Tuncer PB, Sarıozkan S, Ulutas PA, Coyan K, Baspınar N, et al. Effects of hypotaurine, cysteamine and aminoacids solution on post-thaw microscopic and oxidative stress parameters of Angora goat semen. Res Vet Sci. 2009;87(3):468-472.
6. Deana R, Rigoni F, Bonaga G, Andrighetto I, Bonsembiante M, Siliprandi N. Effect of carnitine on functions of bovine spermatozoa. In: Proceedings of the 10th International Congress on Animal Reproduction and Artificial Insemination; 1984 Jun 10-14; UrbanaChampaign, IL. Urbana-Champaign: University of Illinois; 1984.
7. El-Raey M, Ma B, Abdel-Ghani MA, Zeidan RM. Lcarnitine enhancing roles on buffalo semen freezability, ultra-structure and fertilizing potentials. Assiut Vet Med J. 2016;62(149):163-173.
8. Gibb Z, Lambourne SR, Quadrelli J, Smith ND, Aitken RJ. L-carnitine and pyruvate are prosurvival factors during the storage of stallion spermatozoa at room temperature. Biol Reprod. 2015;93(4):104.
9. Griveau JF, Dumont E, Renard P, Callegari JP, Le Lannou D. Reactive oxygen species, lipid peroxidation and enzymatic defence systems in human spermatozoa. Reproduction. 1995;103(1):17-26.
10. Gulcin I. Antioxidant and antiradical activities of Lcarnitine. Life Sci. 2006;78(8):803-811.
11. Hussein YS. Cryoprotective efficacy of local Friesian bull's spermatozoa using tris-extender enriched with natural or synthetic antioxidants. Egypt J Anim Prod. 2018;55(1):1-6.
12. Kerner J, Hoppel C. Fatty acid import into mitochondria. Biochim Biophys Acta Mol Cell Biol Lipids. 2000;1486(1):1-17.
13. Lenzi A, Lombardo F, Gandini L, Dondero F. Metabolism and action of L-carnitine: its possible role in sperm tail function. Arch Ital Urol Nefrol Androl. 1992;64(2):187-196.
14. Longobardi V, Salzano A, Campanile G, Marrone R, Palumbo F, Vitiello M, et al. Carnitine supplementation decreases capacitation-like changes of frozen-thawed buffalo spermatozoa. Theriogenology. 2017;88:236-243.
15. Ma B, Ibrahim E, Desouki RM, Hassan MS, Hussein HM. Effects of L-Carnitine on Morphology and Antioxidant Enzymes and DNA integrity of cryopreserved Buffalo spermatozoa. Assiut Vet Med J. 2017;63(152):173-182.
16. Patel MP, Chaudhari CF, Sutaria TV, Chaudhari RK, Joshi RD, Patel KG, et al. CASA based assessment of post-thaw sperm motion traits as fertility indicators in Mehsana buffalo bulls. Int J Vet Sci Anim Husb. 2026;11(1):380-384.
17. Patel MP, Chaudhari CF, Sutaria TV, Chaudhari RK, Joshi RD, Patel KG, et al. Seminal characteristics and oxidative stress in high-fertility versus low-fertility Mehsana buffalo bulls. Int J Vet Sci Anim Husb. 2024;9(2):695-698.
18. Sharma RK, Agarwal A. Role of reactive oxygen species in male infertility. Urology. 1996;48(6):835-850.
19. Silvestre MA, Yániz JL, Peña FJ, Santolaria P, CastellóRuiz M. Role of antioxidants in cooled liquid storage of mammal spermatozoa. Antioxidants. 2021;10(7):1096.
20. Surai PF. Antioxidant action of carnitine: molecular mechanisms and practical applications. EC Vet Sci. 2015;2(1):66-84.
21. Vaghasiya YH, Chaudhari CF, Joshi RD, Chaudhari RK, Sutaria TV, Chaudhary BG, et al. Assessing the impact of L-Carnitine as a Semen additive on semen parameters and oxidative stress markers in Kankrej bulls. Int J Vet Sci Anim Husb. 2024;9(2 Spec No):155-159.
22. Vaz FM, Wanders RJ. Carnitine biosynthesis in mammals. Biochem J. 2002;361(3):417-429.
23. Wafa WM, El-Nagar HA, Hussein YS, Saeed AM. Effect of different antioxidant sources added to buffalo semen extender during cryopreservation on freezability and fertility of buffalo spermatozoa. J Anim Health Prod. 2021;9(3):222-228.